1. Field of the Invention
This invention relates to a method for detecting bacterial spores using a combination of gas chromatography and mass spectrometry.
2. Description of Related Art
The detection of bacterial spores, or endospores, is useful for a wide variety of purposes. For example, potential biological agents used in warfare and by terrorists include spore-producing bacteria that cause diseases, such as anthrax, botulism, gas gangrene, and tetanus. The detection of spores of such bacteria can also be important in determining and implementing appropriate countermeasures against such agents. In agricultural technologies, spore-producing bacteria, such as Bacillus thuringiensis, are often used as insecticides. Spores in soil or plant samples can be monitored to ensure that the bacteria population is sufficiently high to be effective against insect pests. In the sanitation and hygiene fields, the detection of bacteria spores can be useful to monitor indoor environments, water quality, and food quality. In addition, the detection of nonviable bacterial spores can be useful to paleontologists.
Bacterial endospore concentrations are not easily determined with conventional techniques. Among the primary conventional techniques are microscopy and plate culture counting, both of which are extremely slow and tedious to perform.
U.S. Pat. No. 5,876,960 to Rosen discloses methods for detecting and quantifying bacterial spores in a sample. The method utilizes a lanthanide combined with a medium to be tested for endospore content. The lanthanide reacts with calcium dipicolinate, which is present in bacterial spores, to produce a lanthanide chelate, having distinctive absorbance and emission spectrums, which are detected using photoluminescence. This method is limited, however, as it is difficult to access dipicolinic acid because the endospore casing is difficult to penetrate.
Beverly et al., “Analysis of Dipicolinic acid in Bacterial Spores by Electron Monochromator-Mass Spectrometry,” 47th ASMS Conference on Mass Spectrometry and Allied Topics, 1999 discloses a method of measuring dipicolinic acid in Bacillus cereus in which dried bacteria was directly introduced into a mass spectrometer and heated to 400° C. Bacteria spores typically contain from 7 to 14 percent by weight dipicolinic acid or salts thereof. Although the method was able to detect dipicolinic acid, the endospore casings were not reliably opened, making the results less than quantitative.
It would be desirable to develop a method for detecting bacterial spores that does not rely on time consuming and tedious microscopy and plate culture methods. Such a method should reliably and predictably open endospore casings so that the dipicolinic acid within can be accessed and measured as a surrogate for the quantity of bacterial spores present in a sample.